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Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions.

Lynch CD, Gauthier NC, Biais N, Lazar AM, Roca-Cusachs P, Yu CH, Sheetz MP - Mol. Biol. Cell (2011)

Bottom Line: Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules.Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion.Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB(-/-) mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA(-/-) MEFs, but not FlnB(-/-) MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

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FlnA is the major Fln isoform involved in endoplasmic spreading defects. (A) Representative montages of single FlnA+/+ and FlnA–/– MEFs spread for 25 min on FN (red dotted lines demarcate endoplasm regions). (B) Averaged normalized endoplasm areas for each cell line represented in A. Data shown are mean ± SEM (n = 17). Differences were significant throughout the time course (p < 0.001, two-way repeated measures ANOVA). (C) FlnA–/– MEF spread on FN-coated glass for 50 min. Scale = 20 μm. (D) Montage of stress fiber demarcated in C. After stress fiber breakage, the endoplasm is contracted rearward as indicated by red dotted line. (E) Enlarged images demarcated in A. Gaps are seen in ii., with a normal, coherent ectoplasm shown in i. for comparison. (F) Kymographs from lines seen in E. Gaps begin to appear 15 min after the initiation of spreading and persist throughout. Scale: 10 μm and 10 min.
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Figure 6: FlnA is the major Fln isoform involved in endoplasmic spreading defects. (A) Representative montages of single FlnA+/+ and FlnA–/– MEFs spread for 25 min on FN (red dotted lines demarcate endoplasm regions). (B) Averaged normalized endoplasm areas for each cell line represented in A. Data shown are mean ± SEM (n = 17). Differences were significant throughout the time course (p < 0.001, two-way repeated measures ANOVA). (C) FlnA–/– MEF spread on FN-coated glass for 50 min. Scale = 20 μm. (D) Montage of stress fiber demarcated in C. After stress fiber breakage, the endoplasm is contracted rearward as indicated by red dotted line. (E) Enlarged images demarcated in A. Gaps are seen in ii., with a normal, coherent ectoplasm shown in i. for comparison. (F) Kymographs from lines seen in E. Gaps begin to appear 15 min after the initiation of spreading and persist throughout. Scale: 10 μm and 10 min.

Mentions: Given the dramatic motility defects in Fln-depleted MEFs, we asked whether these phenotypes could be attributed to either FlnA or FlnB. FlnA–/– MEFs exhibited a weaker ER spreading deficiency than did Fln-depleted MEFs (Figure 6A) (Supplemental Movie 5 for FlnA+/+, Supplemental Movie 6 for FlnA–/–) whereas FlnB–/– MEFs appeared similar to controls (Figure 1B). Averaged normalized ER areas of FlnA–/– (Figure 6B) but not FlnB–/– MEFs, were significantly decreased when compared with controls. Also, although FlnA–/– MEFs form stress fibers late in spreading, it was common for them to experience a contraction of the endoplasm that was not seen in controls or Fln-depleted MEFs. This contraction was often associated with stress fiber breakage and the appearance of gaps at the endoplasm boundary of FlnA–/– MEFs (Figure 6C and Supplemental Movie 7), suggesting that the cytoskeleton network was formed but was not able to sustain force at the endoplasm boundary. Stress fiber breakage was correlated with endoplasm retraction (Figure 6D), and gaps in the cytoplasm were observed at the endoplasm boundary of FlnA–/– MEFs (Figure 6E) during the contractile phase of spreading (Figure 6F).


Filamin depletion blocks endoplasmic spreading and destabilizes force-bearing adhesions.

Lynch CD, Gauthier NC, Biais N, Lazar AM, Roca-Cusachs P, Yu CH, Sheetz MP - Mol. Biol. Cell (2011)

FlnA is the major Fln isoform involved in endoplasmic spreading defects. (A) Representative montages of single FlnA+/+ and FlnA–/– MEFs spread for 25 min on FN (red dotted lines demarcate endoplasm regions). (B) Averaged normalized endoplasm areas for each cell line represented in A. Data shown are mean ± SEM (n = 17). Differences were significant throughout the time course (p < 0.001, two-way repeated measures ANOVA). (C) FlnA–/– MEF spread on FN-coated glass for 50 min. Scale = 20 μm. (D) Montage of stress fiber demarcated in C. After stress fiber breakage, the endoplasm is contracted rearward as indicated by red dotted line. (E) Enlarged images demarcated in A. Gaps are seen in ii., with a normal, coherent ectoplasm shown in i. for comparison. (F) Kymographs from lines seen in E. Gaps begin to appear 15 min after the initiation of spreading and persist throughout. Scale: 10 μm and 10 min.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 6: FlnA is the major Fln isoform involved in endoplasmic spreading defects. (A) Representative montages of single FlnA+/+ and FlnA–/– MEFs spread for 25 min on FN (red dotted lines demarcate endoplasm regions). (B) Averaged normalized endoplasm areas for each cell line represented in A. Data shown are mean ± SEM (n = 17). Differences were significant throughout the time course (p < 0.001, two-way repeated measures ANOVA). (C) FlnA–/– MEF spread on FN-coated glass for 50 min. Scale = 20 μm. (D) Montage of stress fiber demarcated in C. After stress fiber breakage, the endoplasm is contracted rearward as indicated by red dotted line. (E) Enlarged images demarcated in A. Gaps are seen in ii., with a normal, coherent ectoplasm shown in i. for comparison. (F) Kymographs from lines seen in E. Gaps begin to appear 15 min after the initiation of spreading and persist throughout. Scale: 10 μm and 10 min.
Mentions: Given the dramatic motility defects in Fln-depleted MEFs, we asked whether these phenotypes could be attributed to either FlnA or FlnB. FlnA–/– MEFs exhibited a weaker ER spreading deficiency than did Fln-depleted MEFs (Figure 6A) (Supplemental Movie 5 for FlnA+/+, Supplemental Movie 6 for FlnA–/–) whereas FlnB–/– MEFs appeared similar to controls (Figure 1B). Averaged normalized ER areas of FlnA–/– (Figure 6B) but not FlnB–/– MEFs, were significantly decreased when compared with controls. Also, although FlnA–/– MEFs form stress fibers late in spreading, it was common for them to experience a contraction of the endoplasm that was not seen in controls or Fln-depleted MEFs. This contraction was often associated with stress fiber breakage and the appearance of gaps at the endoplasm boundary of FlnA–/– MEFs (Figure 6C and Supplemental Movie 7), suggesting that the cytoskeleton network was formed but was not able to sustain force at the endoplasm boundary. Stress fiber breakage was correlated with endoplasm retraction (Figure 6D), and gaps in the cytoplasm were observed at the endoplasm boundary of FlnA–/– MEFs (Figure 6E) during the contractile phase of spreading (Figure 6F).

Bottom Line: Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules.Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion.Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Columbia University, New York, NY 10027, USA.

ABSTRACT
Cell motility is an essential process that depends on a coherent, cross-linked actin cytoskeleton that physically coordinates the actions of numerous structural and signaling molecules. The actin cross-linking protein, filamin (Fln), has been implicated in the support of three-dimensional cortical actin networks capable of both maintaining cellular integrity and withstanding large forces. Although numerous studies have examined cells lacking one of the multiple Fln isoforms, compensatory mechanisms can mask novel phenotypes only observable by further Fln depletion. Indeed, shRNA-mediated knockdown of FlnA in FlnB(-/-) mouse embryonic fibroblasts (MEFs) causes a novel endoplasmic spreading deficiency as detected by endoplasmic reticulum markers. Microtubule (MT) extension rates are also decreased but not by peripheral actin flow, because this is also decreased in the Fln-depleted system. Additionally, Fln-depleted MEFs exhibit decreased adhesion stability that appears in increased ruffling of the cell edge, reduced adhesion size, transient traction forces, and decreased stress fibers. FlnA(-/-) MEFs, but not FlnB(-/-) MEFs, also show a moderate defect in endoplasm spreading, characterized by initial extension followed by abrupt retractions and stress fiber fracture. FlnA localizes to actin linkages surrounding the endoplasm, adhesions, and stress fibers. Thus we suggest that Flns have a major role in the maintenance of actin-based mechanical linkages that enable endoplasmic spreading and MT extension as well as sustained traction forces and mature focal adhesions.

Show MeSH
Related in: MedlinePlus